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Sensitivity of Carrot Cell Cultures and RNA Polymerase II to Amatoxins ¹

Evidence for the Inactivation of 6'-Hydroxyamatoxins

Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida 32611

Protoplast and cell suspension cultures of Daucus carota L. were evaluated for their sensitivity toward the three amatoxin derivatives, -amanitin, 6'-deoxy--amanitin, and 6'-O-methyl--amanitin using inhibition of DNA synthesis to measure cell viability. Protoplasts appeared approximately 10-fold more refractory than suspension cells and -amanitin was much less effective than the other two amatoxins, even though K_i values for isolated RNA polymerase II were similar (4-5 nanomolar). Additional studies evaluating the recoveries of all three amatoxins from cell suspension supernates indicate one basis for these differences to be the selective degradation of -amanitin. A mechanism involving the activation of the hydroxyindole moiety of the -amanitin is thus invoked to explain these differences and we postulate the involvement of plant oxidases in this role.